多药缓释抗菌抗炎支架的制备与性能分析
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摘要
背景:处理创伤局部软组织感染时,全身口服抗生素局部药物浓度较低,无法满足创伤局部抑菌的需要,局部喷洒抗生素粉末虽能临时获得一过性的高浓度,但难以取得较满意的抑菌效果,且高浓度药物对局部组织亦存在一定的破坏效应。目的:开发一种能承载多药并有控释特性的载药缓释支架,用于创伤局部的抗菌抗炎治疗。方法:将左氧氟沙星、替硝唑及甲基强的松龙混合溶液与介孔氧化硅纳米颗粒在负压吸引装置中混合,制备介孔氧化硅纳米颗粒载药微球;根据预先设计好的CAD模型,采用三维打印机交替打印介孔氧化硅纳米颗粒载药微球与聚乳酸-羟基乙酸共聚物层层叠加的多孔网络结构,最终得到膜状载药复合支架(实验组),同时采用药物浸滞法制备载3种药物的磷酸钙多孔陶瓷支架,作为对照,检测2组支架的载药率。将2组支架分别于成纤维细胞共培养,采用CCK-8法检测细胞增殖,评价支架的细胞毒性。将2组支架分别浸泡于PBS中,检测支架内药物体外释放规律。将2组支架分别敷于新西兰大白兔(解放军海军军医大学动物实验中心提供)软组织损伤部位,术后不同时间点检测肝肾功能及血样、创伤周围组织中的药物浓度。结果与结论:①实验组支架中左氧氟沙星、替硝唑及甲基强的松龙的载药率分别为31.21%,22.14%和23.58%,对照组支架3种药物的载药率分别为19.44%,11.14%和9.32%;②2种载药支架上不同时间点的细胞增殖率均在80%以上,细胞毒性为1级;③在体外,对照组支架中的3种药物在第2周基本释放完全,实验组支架中左氧氟沙星释放可达10周,替硝唑及甲基强的松龙释放可达8周;④在体内,实验组血样与局部组织中的3种药物释放时间均长于对照组,药物有效释放时间可达10周以上;实验组支架对兔肝功能存在一过性的影响;⑤结果表明,采用三维打印技术成功制备的多药缓释支架,有望在创伤局部实现联合、高量及持续的局部抗菌及抗炎作用,有效应用于各类局部创伤的辅助治疗。
BACKGROUND: When handling the local infection of the soft tissue of the wound, systemic oral antibiotics due to local concentration is low,cannot satisfy the bacteriostatic needs of local trauma. Although local antibiotics spraying powder can get a temporary high concentration, it is difficult to obtain satisfactory antibacterial effect, and high concentrations of drugs play destructive effect on local tissues.OBJECTIVE: To develop a drug-loaded sustained-release scaffold that can carry multiple drugs, so as to be used for the local antibacterial and anti-inflammatory treatment of trauma.METHODS: Mesoporous silica nanoparticles loaded with drug microspheres were prepared by mixing levofloxacin, tinidazole and methylprednisone solution with mesoporous silica nanoparticles in a negative pressure suction device. According to the designed CAD model in advance, the layer upon layer porous network structure of mesoporous silica nanoparticles loaded with drug microspheres and poly lactide-glycolide acid was alternately printed by three-dimensional printer. Finally, the membrane drug-loaded composite scaffold was obtained(experimental group). Meanwhile, calcium phosphate porous ceramic scaffolds loaded with three drugs were served as control group.The drug loading rate of scaffolds was detected. The two kinds of scaffolds were respectively co-cultured with fibroblasts. The cell proliferation was detected by cell counting-kit 8 assay to evaluate the cytotoxicity of scaffolds. The two scaffolds were immersed in PBS, respectively, to detect the drug release rule in vitro. These scaffolds were applied to the soft tissue injury sites of New Zealand white rabbits(provided by the Laboratory Animal Center of the Navy Medical University). The liver and kidney function, blood samples and drug concentrations in the surrounding tissues were detected at postoperative different time points.RESULTS AND CONCLUSION:(1) The drug loading rates of levofloxacin, tinidazole and methylprednisolone in the scaffold of the experimental group were 31.21%, 22.14% and 23.58%, respectively. The drug loading rates of the three drugs in the scaffold of the control group were 19.44%, 11.14% and 9.32%, respectively.(2) The cell proliferation rates of in the two kinds of scaffolds at different time points were more than 80%, with grade 1 cytotoxicity.(3) In vitro, the release of the three drugs in the scaffold of the control group was basically complete at 2 weeks, the release of levofloxacin in the scaffold of the experimental group was up to 10 weeks, and the release of tinidazole and methylprednisolone in the scaffold of the experimental group was up to 8 weeks.(4) In vivo, the release time of the three drugs in blood samples and local tissues in the experimental group was longer than that in the control group, and the effective release time of the drugs was more than 10 weeks. The scaffold of the experimental group had transient effect on the liver function of rabbits(5) In summary, the multi-drug sustained-release scaffolds are successfully prepared by three-dimensional printing technology, which is expected to achieve combined, high and continuous local antibacterial and anti-inflammatory effects in the local wound, and be effectively applied in the adjuvant treatment of various local wounds.
BACKGROUND: When handling the local infection of the soft tissue of the wound, systemic oral antibiotics due to local concentration is low,cannot satisfy the bacteriostatic needs of local trauma. Although local antibiotics spraying powder can get a temporary high concentration, it is difficult to obtain satisfactory antibacterial effect, and high concentrations of drugs play destructive effect on local tissues.OBJECTIVE: To develop a drug-loaded sustained-release scaffold that can carry multiple drugs, so as to be used for the local antibacterial and anti-inflammatory treatment of trauma.METHODS: Mesoporous silica nanoparticles loaded with drug microspheres were prepared by mixing levofloxacin, tinidazole and methylprednisone solution with mesoporous silica nanoparticles in a negative pressure suction device. According to the designed CAD model in advance, the layer upon layer porous network structure of mesoporous silica nanoparticles loaded with drug microspheres and poly lactide-glycolide acid was alternately printed by three-dimensional printer. Finally, the membrane drug-loaded composite scaffold was obtained(experimental group). Meanwhile, calcium phosphate porous ceramic scaffolds loaded with three drugs were served as control group.The drug loading rate of scaffolds was detected. The two kinds of scaffolds were respectively co-cultured with fibroblasts. The cell proliferation was detected by cell counting-kit 8 assay to evaluate the cytotoxicity of scaffolds. The two scaffolds were immersed in PBS, respectively, to detect the drug release rule in vitro. These scaffolds were applied to the soft tissue injury sites of New Zealand white rabbits(provided by the Laboratory Animal Center of the Navy Medical University). The liver and kidney function, blood samples and drug concentrations in the surrounding tissues were detected at postoperative different time points.RESULTS AND CONCLUSION:(1) The drug loading rates of levofloxacin, tinidazole and methylprednisolone in the scaffold of the experimental group were 31.21%, 22.14% and 23.58%, respectively. The drug loading rates of the three drugs in the scaffold of the control group were 19.44%, 11.14% and 9.32%, respectively.(2) The cell proliferation rates of in the two kinds of scaffolds at different time points were more than 80%, with grade 1 cytotoxicity.(3) In vitro, the release of the three drugs in the scaffold of the control group was basically complete at 2 weeks, the release of levofloxacin in the scaffold of the experimental group was up to 10 weeks, and the release of tinidazole and methylprednisolone in the scaffold of the experimental group was up to 8 weeks.(4) In vivo, the release time of the three drugs in blood samples and local tissues in the experimental group was longer than that in the control group, and the effective release time of the drugs was more than 10 weeks. The scaffold of the experimental group had transient effect on the liver function of rabbits(5) In summary, the multi-drug sustained-release scaffolds are successfully prepared by three-dimensional printing technology, which is expected to achieve combined, high and continuous local antibacterial and anti-inflammatory effects in the local wound, and be effectively applied in the adjuvant treatment of various local wounds.
引文
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[4]陈萍,刘丁.创伤患者的医院感染危险因素研究[J].中华医院感染学杂志,1999,9(2):74-76.
[5]陈慧华,陈海华,林福秀,等.创伤性骨折术后切口感染的影响因素及预防对策[J].中华医院感染学杂志,2013,23(14):3395-3396.
[6]方丁,吉璐宏,陈盛,等.四肢开放性创伤后机体免疫应激及创面病原菌变化的研究[J].中国实验诊断学,2017,21(7):1230-1232.
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[12]沈宏亮,钱方,范列英.左氧氟沙星壳聚糖缓释微球对海水浸泡创伤的初期治疗结果[J].外科理论与实践,2005,10(1):73-76.
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[14]Yang XM,Cheng YY,Zhang ZL,et al.Role of Methylprednisolone in Treatment of Spinal Cord Injured with Bone Marrow Mesenchymal Stem Cells Transplantation in Rats and Its Effect on the Expressions of Tumor Necrosis Factor-αand Interleukin-1β.Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2017;39(5):615-622.
[15]王玉,韩璐,车顺爱.具有可控分散性的不同粒径氨基酸双功能化介孔二氧化硅纳米颗粒[J].高等学校化学学报,2011,32(3):560-570.
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[22]Radu DR,Lai CY,Jeftinija K,et al.A polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent.J Am Chem Soc.2004;126(41):13216-13217.
[23]李坤.基于三维打印技术的载药抗结核骨支架的构建与特性研究[D].上海:第二军医大学,2016.
[24]中华医学会创伤学分会.中国区域性创伤救治体系建设的专家建议[J].中华外科杂志,2015,53(8):571-573.
[25]张连阳.提升创伤中心救治能力的关键途径[J].中华创伤杂志,2018,34(10):869-871.
[26]吴素英.碱性成纤维细胞生长因子与小儿烧伤创面的愈合[J].中国组织工程研究,2012,16(33):6215-6222.
[27]张琴,宛云英.深圳市2007~2011年突发事件院外急救分析[J].中国急救复苏与灾害医学杂志,2014,9(10):891-893,914.
[28]于开庆.突发意外、交通事故致多人重症多发伤复合外伤649例院前转运救治分析[J].中国社区医师(医学专业),2012,14(30):170.
[29]抗菌感染新途径[J].中华危重病急救医学,2015,27(11):884-884.
[30]李芳春.负压封闭式引流术(VSD)在四肢创伤合并皮肤软组织大面积缺损中的临床应用效果[J].世界最新医学信息文摘(连续型电子期刊),2015,15(78):97.
[31]李东镖,武志坚,王霄虎.封闭负压引流技术治疗25例骨科创伤感染临床疗效分析[J].吉林医学,2011,32(36):7703-7704.
[32]胡艳,余江,郑慧新.皮肤创面亲水性纤维含银敷料在四肢软组织创伤合并感染护理中应用的价值[J].检验医学与临床,2017,14(Z1):333-335.
[33]徐溪,莫乃新,陈文美,等.亲水性纤维含银敷料与磺胺嘧啶银霜治疗深Ⅱ度烧伤的疗效观察[J].中国医院用药评价与分析,2016,16(4):486-488.
[34]Victor SP,Sharma CP.Poly methacrylic acid modified CDHAnanocomposites as potential p H responsive drug delivery vehicles.Colloids Surf B Biointerfaces.2013;108:219-228.
[35]Francis L,Meng D,Knowles JC,et al.Multi-functional P(3HB)microsphere/45S5 Bioglass-based composite scaffolds for bone tissue engineering.Acta Biomater.2010;6(7):2773-2286.
[36]Dutt M,Khuller GK.Sustained release of isoniazid from a single injectable dose of poly(DL-lactide-co-glycolide)microparticles as a therapeutic approach towards tuberculosis.Int J Antimicrob Agents.2001;17(2):115-122.
[37]Vacanti NM,Cheng H,Hill PS,et al.Localized delivery of dexamethasone from electrospun fibers reduces the foreign body response.Biomacromolecules.2012;13(10):3031-3038.
[38]Zhang J,Wang C,Wang J,et al.In vivo drug release and antibacterial properties of vancomycin loaded hydroxyapatite/chitosan composite.Drug Deliv.2012;19(5):264-269.
[39]Chen C,Wang H,Zhu G,et al.Three-dimensional poly lactic-co-glycolic acid scaffold containing autologous platelet-rich plasma supports keloid fibroblast growth and contributes to keloid formation in a nude mouse model.J Dermatol Sci.2018;89(1):67-76.
[40]Jonard B,Dean E.Posttraumatic Reconstruction of the Foot and Ankle in the Face of Active Infection.Orthop Clin North Am.2017;48(2):249-258.
[2]刘中民.改善急救模式提高创伤救治水平[J].中华急诊医学杂志,2002,11(2)79-80.
[3]都定元,高劲谋.严重交通伤与坠落伤救治结局比较和创伤急救模式探讨[J].中华创伤杂志,2000,16(1):46-48.
[4]陈萍,刘丁.创伤患者的医院感染危险因素研究[J].中华医院感染学杂志,1999,9(2):74-76.
[5]陈慧华,陈海华,林福秀,等.创伤性骨折术后切口感染的影响因素及预防对策[J].中华医院感染学杂志,2013,23(14):3395-3396.
[6]方丁,吉璐宏,陈盛,等.四肢开放性创伤后机体免疫应激及创面病原菌变化的研究[J].中国实验诊断学,2017,21(7):1230-1232.
[7]冯雪.早期局部应用抗生素药物对颌面部创伤感染的研究[J].养生保健指南:医药研究,2015,14(11):7.
[8]张永民,施忠民,金国华,等.局部载体抗生素治疗四肢创伤后感染的疗效观察[J].全科医学临床与教育,2006,4(4):287-288.
[9]芮清龙,魏善和,李修英.开放性创伤伤口感染抗生素预防和治疗[J].中华腹部疾病杂志,2006,6(8):593-595.
[10]孙海晨.问题2:创伤感染应用抗生素的基本原则是什么?[J].创伤外科杂志,2014,16(3):228-228.
[11]廖小萍.抗生素的应用表现及耐药性分析[J].中国医药指南,2014,12(29):31-31,33.
[12]沈宏亮,钱方,范列英.左氧氟沙星壳聚糖缓释微球对海水浸泡创伤的初期治疗结果[J].外科理论与实践,2005,10(1):73-76.
[13]孔熙.左氧氟沙星联合替硝唑治疗盆腔炎疗效观察[J].现代中西医结合杂志,2014,23(14):1536-1537.
[14]Yang XM,Cheng YY,Zhang ZL,et al.Role of Methylprednisolone in Treatment of Spinal Cord Injured with Bone Marrow Mesenchymal Stem Cells Transplantation in Rats and Its Effect on the Expressions of Tumor Necrosis Factor-αand Interleukin-1β.Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2017;39(5):615-622.
[15]王玉,韩璐,车顺爱.具有可控分散性的不同粒径氨基酸双功能化介孔二氧化硅纳米颗粒[J].高等学校化学学报,2011,32(3):560-570.
[16]Zhu M,Wang H,Liu J,et al.A mesoporous silica nanoparticulate/β-TCP/BG composite drug delivery system for osteoarticular tuberculosis therapy.Biomaterials.2011;32(7):1986-1995.
[17]王会学.局部植入治疗骨与关节结核药物缓释体的制备与特性研究[D].上海:第二军医大学,2011.
[18]刘许,宋阳.用于3D打印的生物相容性高分子材料[J].合成树脂及塑料,2015,32(4):96-99,102.
[19]Butscher A,Bohner M,Hofmann S,et al.Structural and material approaches to bone tissue engineering in powder-based three-dimensional printing.Acta Biomater.2011;7(3):907-920.
[20]Chen M,Le DQ,Hein S,et al.Fabrication and characterization of a rapid prototyped tissue engineering scaffold with embedded multicomponent matrix for controlled drug release.Int J Nanomedicine.2012;7:4285-4297.
[21]伍卫刚,郑启新,郭晓东.利福平-异烟肼-控释型载药人工骨的实验研究[J].中国生物医学工程学报,2010,29(1):137-143.
[22]Radu DR,Lai CY,Jeftinija K,et al.A polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent.J Am Chem Soc.2004;126(41):13216-13217.
[23]李坤.基于三维打印技术的载药抗结核骨支架的构建与特性研究[D].上海:第二军医大学,2016.
[24]中华医学会创伤学分会.中国区域性创伤救治体系建设的专家建议[J].中华外科杂志,2015,53(8):571-573.
[25]张连阳.提升创伤中心救治能力的关键途径[J].中华创伤杂志,2018,34(10):869-871.
[26]吴素英.碱性成纤维细胞生长因子与小儿烧伤创面的愈合[J].中国组织工程研究,2012,16(33):6215-6222.
[27]张琴,宛云英.深圳市2007~2011年突发事件院外急救分析[J].中国急救复苏与灾害医学杂志,2014,9(10):891-893,914.
[28]于开庆.突发意外、交通事故致多人重症多发伤复合外伤649例院前转运救治分析[J].中国社区医师(医学专业),2012,14(30):170.
[29]抗菌感染新途径[J].中华危重病急救医学,2015,27(11):884-884.
[30]李芳春.负压封闭式引流术(VSD)在四肢创伤合并皮肤软组织大面积缺损中的临床应用效果[J].世界最新医学信息文摘(连续型电子期刊),2015,15(78):97.
[31]李东镖,武志坚,王霄虎.封闭负压引流技术治疗25例骨科创伤感染临床疗效分析[J].吉林医学,2011,32(36):7703-7704.
[32]胡艳,余江,郑慧新.皮肤创面亲水性纤维含银敷料在四肢软组织创伤合并感染护理中应用的价值[J].检验医学与临床,2017,14(Z1):333-335.
[33]徐溪,莫乃新,陈文美,等.亲水性纤维含银敷料与磺胺嘧啶银霜治疗深Ⅱ度烧伤的疗效观察[J].中国医院用药评价与分析,2016,16(4):486-488.
[34]Victor SP,Sharma CP.Poly methacrylic acid modified CDHAnanocomposites as potential p H responsive drug delivery vehicles.Colloids Surf B Biointerfaces.2013;108:219-228.
[35]Francis L,Meng D,Knowles JC,et al.Multi-functional P(3HB)microsphere/45S5 Bioglass-based composite scaffolds for bone tissue engineering.Acta Biomater.2010;6(7):2773-2286.
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